Alkalinity and groundwater CO2 have increased linearly from 1991–2017 at the Konza Prairie Biological Station (KPBS), a tallgrass prairie research site in northeastern Kansas. The projected increase in groundwater alkalinity (as HCO3-) and CO2 based on an earlier trend was confirmed in 2016, with predictions nearly equal to recent values (e.g., 408 ppm vs 410 ppm as HCO3-, respectively). Both the water balance and groundwater CO2 trends within the study watershed could be impacted by long-term changes in land use and climate: 1) encroachment of woody vegetation (1983–2012) as a result of the 4-year fire return interval, 2) re-introduction of bison (phased in, 1994–2006), 3) increases in air temperature, and 4) changes in precipitation patterns. If only linear processes are driving the observed water chemistry changes, then the linear increase in air temperature (1983–2017) that stimulates soil respiration may be the most likely factor enhancing groundwater HCO3- and CO2, as air temperature has risen ~1 to 1.4°C over 34 years. If groundwater chemistry is driven by more threshold behaviour, woody encroachment, which was linear but in three distinct phases, may drive groundwater chemistry. The ~2 to 3‰ decrease in the discontinuous δ13C data in the groundwater-dominated stream suggests enhanced inputs of microbially-respired labile carbon, CO2 sourced from C3 (woody vegetation), or a combination of the two.